Development of Compact CRISPR Editors for Multiplexed Editing of Complex Gene Networks Abstract O-Linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide modification of nucleocytoplasmic proteins that serves as an important chemical code in regulation and signaling processes. Specifically, O-GlcNAc modification of transcription factors plays a profound role in transcription regulation. However, previous efforts in studying the dynamic O-GlcNAc code in a complex gene network were hampered by the inability to perform protein-specific and locus-specific O-GlcNAc modification of transcription factors. The objective of our parental 1DP2HG011027-01 project is to develop compact, multiplexable CRISPR editors to allow the genomic editing at transcriptional, epigenetic, and post- translational levels. In line with this parental project, we now request an administrative supplement with the objective to extend our CRISPR editors to enable locus-specific engineering of the O-GlcNAc modification of transcription factors and study how dynamic O-GlcNAc codes regulate transcription and gene expression. To achieve this objective, we will collaborate with Professor Christina Woo?s group at Harvard University and adopt the tools developed by their 1U01CA242098-01 project in our efforts to develop CRISPR editors for engineering transcription factor-specific O-GlcNAc modifications. By fusing O- GlcNAc transferase (OGT) and O-GlcNAcase (OGA) with a nanobody, Woo?s O-GlcNAc engineering constructs can be efficiently recruited by Cas9 proteins tagged by the epitope of the nanobody. We will investigate the optimal distance between the CIRPSR-mediated OGT/OGA binding site and the target transcription factor using specifically designed reporter constructs that translate the transcription factor activity into reporter signal in vivo. Furthermore, we will integrate the RNA aptamer technology developed in our parental project into proximity-directed O-GlcNAc engineering and evolve RNA aptamers in vitro and in vivo that can recruit endogenous OGT and OGA to the CRISPR editors for reprogramming O-GlcNAc codes on target transcription factors. The proposed research will lead to a novel method to engineer the O-GlcNAc code of transcription factors and allow us to study the dynamic interplay of O-GlcNAc modifications at different transcription factors in gene networks.